Simultaneous determination of six C21 steroids of Xiao-Ai-Ping injection in rat plasma by LC-MS/MS.

Xiao-Ai-Ping injection (XAPI) is a traditional Chinese medicine that has been widely used to treat cancer. Modern pharmacological studies have demonstrated that C21 steroids are the main active compounds in XAPI. In this study, a sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated the first time for simultanenous determination of three isomeric pregnane genins (17β-tenacigenin B, tenacigenin B and tenacigenin A) and their corresponding glycosides (tenacigenoside A, tenacissoside F and marsdenoside I) from XAPI in rat plasma. A simple liquid-liquid extraction technique was used after the addition of dexamethasone acetate as internal standard. The chromatography separation of analytes was achieved on an Agilent Zorbax Eclipse XDB-C18 column (3.5 µm, 150 × 3 mm i.d.) using methanol-water as mobile phase in a gradient elution program. Detection was performed in multiple reaction monitoring mode using electrospray ionization in the negative ion mode. The method showed satisfactory linearity over a concentration range 5.00-2000.00 ng/mL for tenacigenin B, tenacigenin A, marsdenoside I and tenacissoside F (r(2) > 0.99), 10.00-4000.00 ng/mL for 17β-tenacigenin B and tenacigenoside A (r(2) > 0.99). Intra- and inter-day precisions (valued as relative standard deviation) were <9.00% and accuracies (as relative error) in the range -6.31 to 7.23%. Finally, this validated method was successfully applied to the pharmacokinetic study of XAPI after intravenous administration to rats.